AOAC Guidance on FA Immunoassay Validation (August 2023)

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pilot-scale mixing equipment may also be appropriate, depending on the matrix type. When using either kitchen- or pilot-scale equipment, be aware that mechanical blending very often generates heat when conducted for extended periods. Depending on the extent of heat generated, the food matrix and/or food allergen materials may be impacted if processes are 3.8.3. In some cases, low concentrations are challenging to achieve even with 1-kg batch sizes. In these instances, a higher working concentration of dry blended material can be used to prepare test materials with lower concentrations of the target food allergen. For example, to prepare cookies incurred with milk protein, a working material of 1,000 mg total milk protein/kg wheat flour can be prepared by thoroughly mixing 2.86 g nonfat dry milk (NFDM, 35% protein) in 997.14 g wheat flour. This working material can subsequently be used to prepare batches of cookies of desired concentrations. For example, if a 1-kg batch of cookie dough is to be prepared with a target concentration of 10 mg total milk protein/kg cookie dough, 10 g of the 1,000 mg total milk protein/kg wheat flour working mixture can be substituted for 10 g of the flour in the cookie formulation (10 g of 1,000 mg total milk not adequately controlled.

protein/kg wheat flour + 990 g of cookie dough ingredients).

3.8.4. More examples are available in Taylor et al. 2009. (1)

3.8.5. When developing a new procedure for preparing incurred foods, it is advisable for method developers to evaluate the homogeneity of the material throughout preparation. Use of a testing material with heterogeneous food allergen material distribution likely will deliver higher than expected levels of variance in method performance. An example of beneficial information collection is the analysis of the high concentration working material used to

prepare other materials.

3.8.5.1.

Suggestion for homogeneity testing: Perform 10 extractions and run method as described. Calculate %CV. For quantitative methods, the homogeneity testing %CV

should be less than what is accepted for the method.

3.8.6. In instances with food processing operations that are sufficiently complex as to restrict the number of concentration levels that are feasible to prepare, higher-level incurred samples can be mixed with blank, processed samples to achieve various concentrations. Lowest concentration achieved in this way should not be less than 10% of the concentration of the high-level incurred material. Larger discrepancies require a second incurred matrix to be 1226 4.1. For some sections (5.2.3 and 6.2.2), spiking is the allowed method of sample preparation. Spiked 1227 samples are created by adding the allergenic reference material after the final processing steps 1228 have been completed but prior to extraction. 1229 4.2. Bulk spiking: See preparation steps for solid into solid and liquid into liquid bulk samples as 1230 described in incurred section. Following preparation spiked samples do not need to go through 1231 additional processing steps. 1232 4.3. Test portion spiking 1233 4.3.1. Spiking individual test portions with representative food allergen materials is very difficult and 1234 is not generally advised due to challenges with suspension homogeneity and the small 1235 volumes required. 1236 made at a lower level. 4. Spiked Samples